The environmental pollution by internal secretion-disturbing substances becomes serious problems in the atmosphere, incineration ashes, exhaust gas, drain, foods, marine products, soil and the like, and research and investigation have been made on the influences of the environmental pollution on human being and the other organisms, not only in Japan but also many countries of the world. In particular, it is suspected that dioxins lastingly influence an ecological system of human being and the other organisms. Accordingly, investigation of polluted conditions, elucidation of exposure conditions to human being and the other organisms and of intake routes as well as development of a simple monitoring method which can be used rapidly and at many points in polluted facilities, polluted locations and exhaust sources of dioxins have been carried out.
The term “dioxins” is a comprehensive term including dioxins comprised of 75 kinds of polychlorodibenzo-p-dioxins (PCDDs) and 135 kinds of polychlorodibenzofurans (PCDFs) as well as coplanar polychlorobiphenyls, and therefore, there are many structural isomers (they may be referred to as “dioxin isomers” collectively in the specification). The relative toxicity of each dioxin isomer, when assuming the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) having the highest toxicity among the above isomers is 1, is shown as the Toxic Equivalency Factor (TEF) by the World Health Organization (WHO), and analysis of dioxins in samples is carried out using 7 kinds of PCDDs and 10 kinds of PCDFs having toxicity values. Thus, the actually determined concentration of each isomer is multiplied by the corresponding TEF value, and the sum total of the values obtained for all the isomers is calculated as the 2,3,7,8-TCDD Toxic Equivalent quantity (TEQ). The TEQ values are used as analytical values of the dioxins.
Also, of polychlorobiphenyls (PCBs) previously known as pollutants in the environment, totally 12 kinds of coplanar PCBs including 4 kinds of non-ortho PCBs and 8 kinds of mono-ortho PCBs have been measured as dioxins, because they show biological effects similar to those of dioxins.
Previously, the determination of dioxins has been carried out by measuring the above 17 kinds of dioxin isomers, by high-resolution gas chromatography/mass spectrometry (GC/MS method) using an expensive analytical instrument, and then calculating the TEQ value from the measured values. However, the GC/MS method needs removal of interfering substances contained in samples, and therefore, a multi-stage of complicated cleanup procedures, skilled analyzers and so on. Also, the method needs much time for obtaining and analyzing the data, and therefore, the use of the method is limited to an analysis in a particular analytical facility. On the other hand, due to the recent innovative progress of investigations, there is a tendency to analyze internal secretion-disturbing substances by a biological technique. Thus, attention is paid to a bioassay, a receptor-binding assay based on biological samples, an immunoassay and the like, for the reason that they are suitable for rapid and simple measurement of samples from the environment containing enormous kinds of dioxin isomers, and they have been developed intensively. In application to the analysis of samples from the environment, however, there arise problems such as deviation from analytical values obtained by the official method (GC/MS method) and lack of reproducibility of a measuring system. Accordingly, it is desired to develop a simple analytical method suitable for measurement of samples from the environment.
Recently proposed is an idea that a total dioxin amount (TEQ) is simply determined by measurement of a particular indicator isomer. In a wide range of samples, for example, samples from the environment such as soil, mud, atmosphere, water, exhaust gas and ash, samples from a living body such as mother's milk and blood as well as marine products, foods and the like, it has been proved that the amount of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF), which is one of dioxin isomers each having a predetermined TEF value has a very high correlation with the TEQ value (non-patent reference 1). In view of the report, the present inventors investigated a method of determining dioxins using the indicator isomer as a target, developed recombinant antibodies having a high specificity to 2,3,4,7,8-PeCDF, and filed a patent application directed to the subject matters (Japanese Patent Application No. 2003-091663). However, it was found that an antibody having not only a reactivity with the indicator isomer but also a cross-reactivity with plural dioxin isomers which contribute largely to a TEQ value is needed in order to obtain a measuring method having a higher correlation with samples from the environment, and that the use of the antibody provides measured values having a higher correlation with GC/MS values.
Among biological techniques for analysis of dioxins, an attempt to quantifying the dioxins by an immunochemical technique utilizing an antibody is made, for example, in patent reference 1. Thus, the reference discloses a method for detecting dioxins contained in samples from a living body such as human blood and mother's milk by preparing and using a monoclonal antibody having a high affinity for 2,3,7,8-TCDD. Also, patent reference 2 discloses a method for deducing an amount of dioxins present in samples using a few antibodies having a highest affinity for 2,3,7,8-TCDD and also having a cross-reactivity with the other plural isomers. In these references, however, there is no sufficient description as to monoclonal antibodies recognizing dioxins having five or six chlorine atoms as well as measuring methods using said monoclonal antibodies which methods have a correlation with the total dioxin amount (TEQ) in samples from the environment. Thus, these references fail to describe a concrete practice of a measuring method for samples from the environment.
Furthermore, patent reference 3 discloses a method for determining dioxins in soil or mud using a polyclonal antibody having a high affinity for dioxins having eight chlorine atoms. However, this reference does not show a definite correlation between the values measured using the antibody and the GC/MS values measured by instrumental analysis, for dioxins having eight chlorine atoms in samples.
On the other hand, non-patent reference 2 discloses an antibody which shows a cross-reactivity, when assuming the reactivity with 1,2,3,7,8-PeCDF is 100%, of 39.8% with 1,2,3,7,8,9-HxCDD, of 45.9% with 1,2,3,4,7,8-HxCDF, of 36.6% with 1,2,3,6,7,8-HxCDF, of 42.7% with 1,2,3,7,8,9-HxCDF, and of 43.8% with 2,3,4,6,7,8-HxCDF, among 17 kinds of PCDDs and PCDFs each having a predetermined WHO-TEF value. Thus, this reference discloses an antibody having a broad cross-reactivity with dioxins, which shows about 35% to 45% of a cross-reactivity with all PCDFs having six chlorine atoms and one PCDD having six chlorine atoms and also shows about 10% of a reactivity with the other dioxin isomers each having a TEF value. Also, the reference reports that a certain correlation can be seen between the values measured using said antibody for samples from the environment and the GC/MS values.                Non-patent reference 1: Takasuga et al., 11th Symposium on Environmental Chemistry, Program and Abstracts, p. 136, 2002;        Non-patent reference 2: Fujihira et al., Environmental Solution Technology, Vol. 2, No. 5, p. 63, 2003;        Patent reference 1: JP-A-2002-228660;        Patent reference 2: JP-A-2002-119279;        Patent reference 3: JP-A-2003-098173.        